Improved Model of Proton Pump Crystal Structure Obtained by Interactive Molecular Dynamics Flexible Fitting Expands the Mechanistic Model for Proton Translocation in P-Type ATPases.

Dorota Focht,Tristan I Croll,Poul Nissen,Bjorn P Pedersen

doi:10.3389/fphys.2017.00202

Dorota Focht, Tristan I Croll + Show 2 more

Open Access

https://doi.org/10.3389/fphys.2017.00202

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Abstract

The plasma membrane H+-ATPase is a proton pump of the P-type ATPase family and essential in plants and fungi. It extrudes protons to regulate pH and maintains a strong proton-motive force that energizes e.g., secondary uptake of nutrients. The only crystal structure of a H+-ATPase (AHA2 from Arabidopsis thaliana) was reported in 2007. Here, we present an improved atomic model of AHA2, obtained by a combination of model rebuilding through interactive molecular dynamics flexible fitting (iMDFF) and structural refinement based on the original data, but using up-to-date refinement methods. More detailed map features prompted local corrections of the transmembrane domain, in particular rearrangement of transmembrane helices 7 and 8, and the cytoplasmic N- and P-domains, and the new model shows improved overall quality and reliability scores. The AHA2 structure shows similarity to the Ca2+-ATPase E1 state, and provides a valuable starting point model for structural and functional analysis of proton transport mechanism of P-type H+-ATPases. Specifically, Asp684 protonation associated with phosphorylation and occlusion of the E1P state may result from hydrogen bond interaction with Asn106. A subsequent deprotonation associated with extracellular release in the E2P state may result from an internal salt bridge formation to an Arg655 residue, which in the present E1 state is stabilized in a solvated pocket. A release mechanism based on an in-built counter-cation was also later proposed for Zn2+-ATPase, for which structures have been determined in Zn2+ released E2P-like states with the salt bridge interaction formed.

Highlights

The Arabidopsis thaliana plasma membrane H+-ATPase 2 (AHA2) is a member of the PIII-subtype of the P-type ATPase superfamily
The application of iMDFF environment in refinement of lowresolution protein structures was successfully reported in studies on Human Insulin Receptor Ectodomain (Croll et al, 2016), and we have applied it here to the original, highly anisotropic 3.6 Å resolution crystallographic data obtained from AHA2 crystals (Pedersen et al, 2007)
Structural changes of the revised AHA2 model includes a local rearrangement of transmembrane helices 7 and 8, where ∼1-turn N-terminal register shift is observed, and local changes at the nucleotide binding pocket of the Ndomain

Summary

INTRODUCTION

The Arabidopsis thaliana plasma membrane H+-ATPase 2 (AHA2) is a member of the PIII-subtype of the P-type ATPase superfamily. It pumps protons out of the cell to maintain a steep electrochemical H+ gradient and potential across the plasma membrane (Serrano et al, 1986; Blatt et al, 1987). The H+ATPases are of fundamental importance in plants and fungi, as well as several prokaryotes (Pedersen et al, 2014) They maintain a membrane potential at around −150 mV in plants, even down to −300 mV in fungi, control intracellular H+ homeostasis and extracellular acidification, and potentiate secondary transporters involved in e.g., nutrient uptake (Briskin, 1990). The PIII-type plasma membrane H+-ATPases share a similar fold with the PII subfamily including Na+/K+ATPases, H+/K+-ATPases, and Ca2+-ATPases present in animal cells (Bublitz et al, 2011). The revised model as well as the body of published data since 2007 invites a reiteration of the structure/function relationship, and it allows tentative studies by molecular dynamics simulations in a lipid bilayer environment

MATERIALS AND METHODS

Findings

CONCLUSIONS